Zhang et al. BMC Medical Genetics(2020) 21:235
https://doi.org/10.1186/s12881-020-01162-3

CASE REPORTOpen AccessFirst case report of cerebral folate
deficiency caused by a novel mutation of
FOLR1 gene in a Chinese patient
Ciliu Zhang1, Xiaolu Deng1, Yafei Wen2, Fang He1, Fei Yin1 and Jing Peng1*

Abstract
Background: Cerebral folate deficiency (CFD) is a neurological disease, hallmarked by remarkable low
concentrations of 5-methyltetrahydrofolic acid (5-MTHF) in cerebrospinal fluid (CSF). The primary causes of CFD
include the presence of folate receptor (FR) autoantibodies, defects of FR encoding gene FOLR1, mitochondrial
diseases and congenital abnormalities in folate metabolism.
Case presentation: Here we first present a Chinese male CFD patient whose seizure onset at 2 years old with
convulsive status epilepticus. Magnetic Resonance Imaging (MRI) revealed the development of encephalomalacia,
laminar necrosis in multiple lobes of the brain and cerebellar atrophy. Whole Exome Sequencing (WES) uncovered ahomozygous missense variant of c.524G > T (p.C175F) in FOLR1 gene. Further laboratory tests demonstrated theextremely low level of 5-MTHF in the CSF from this patient, which was attributed to cerebral folate transport
deficiency. Following the intravenous and oral treatment of calcium folinate, the concentrations of 5-MTHF in CSF
were recovered to the normal range and seizure symptoms were relieved as well.
Conclusions: One novel variation of FOLR1 was firstly identified from a Chinese male patient with tonic-clonicseizures, developmental delay, and ataxia. The WES and laboratory results elucidated the etiology of the symptoms.
Clinical outcomes were improved by early diagnosis and proper treatment.
Keywords: Seizures, FOLR1, 5-MTHF, Calcium folinate

BackgroundAs defined in 2004 by Ramaekers and Blau, Cerebral
Folate Deficiency (CFD) encompasses a collection ofneurological syndromes associated with low cerebrospinal
fluid (CSF) concentrations of 5-methyltetrahydrofolate (5MTHF), the normal folate metabolism in nervous system[1]. The circumstantial evidence show that CFD is causedby congenital metabolic disorders and the acquired folicacid deficiency [2, 3]. The most common cause of CFD isthe presence of folate receptor (FR) autoantibodies [4]. In
addition, defects of FR encoding gene FOLR1,* Correspondence: pengjing4346@163.com
1
Xiangya Hospital Central South University, 87 Xiangya Road, Changsha,
Hunan 410008, P.R. China
Full list of author information is available at the end of the article

mitochondrial diseases and congenital abnormalities infolate metabolism could also lead to CFD. FOLR1(OMIM#613068), located in the long arm of chromosome
11, encodes for folate receptor α (FRα) [5]. Binding of FRαwith 5-MTHF in high affinity is essential for the transportation of folate to the brain [6, 7]. Thus, pathogenic variants of FOLR1 could result in brain-specific 5-MTHF
deficiency, and ultimately cause a series of neuropsychiatric symptoms [5]. Normally, the onset of CFD caused bymutated FOLR1 begins from late infancy [8], with the
major clinical manifestations including developmental
retardation, dyskinesia, epileptic seizure, leukodystrophyand slow EEG background activity. In infant patients, 5MTHF concentration in cerebrospinal fluid is also extremely low, but remains normal in peripheral nervous© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate ifchanges were made. The images or other third party material in this article are included in the article's Creative Commonslicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
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Zhang et al. BMC Medical Genetics(2020) 21:235

system, indicating that the cerebral 5-MTHF deficiency isthe underlying mechanism for infant CFD.

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Hospital, Central South University. Informed consent
was obtained from the parents of this proband.
Genetic analysis

Case presentation
Patient

The Chinese patient was born at full term throughnormal vaginal delivery, weighting 3.25 kg that fell in thenormal range of newborn weights. He showed developmental delay and mental retardation since one and halfyears old. He was able to raise his head at 3 months, sit
at 6 ~ 7 months, walk at 24 months, and learn limited
vocabulary at 12 months. The patient was in poor
physical health since he was 2 years old.
At 2 years of age, the patient started to have tonicclonic seizures that lasted for 20–30 min with high fever(39 degrees Celsius) and was initially diagnosed as febrile
seizure at local hospital. The patient was treated with
anticonvulsive medications. The symptoms were relieved
afterwards. However, a similar yet more severe symptoms appeared around age of four. Following similar
treatments, the patient was transferred to anotherhospital to seek out a detailed clinical examination. CT
scan of brain did not detect lesions. The patient was
subsequently diagnosed with viral meningitis treatedwith anti-virus medicine. At the age of 6 years and 9months, the symptom relapsed. The patient manifested
vomiting, headache, tonic-clonic seizure, symptomatic
generalized epilepsy that could last for half an hour. The
patient was intubated and treated with the combinationof antibiotics, phenobarbitone and sodium valproate.
Although the symptoms were relieved to some extent
when the patient took sodium valproate at dose of 0.2 g
Tid (26 mg/kg/d), the etiology of the symptoms
remained unknown. Therefore, at age of 6 years and 11
months, after about 4 years of repeatedly suffering fromstatus epilepticus, the patient was referred to Xiangya
Hospital, Central South University, Hunan Province,
China. The detailed neurological exams showed that thepatient clinically manifested global developmental delay,slurred speech, hyperactivity, sialorrhea, difficulty inmanaging behavior, unsteady gait, hypotonia. His headcircumference was 46 cm (less than the 3rd percentile).
An extensive laboratory and genetic analysis were performed to uncover the etiology of his disease.
We performed brain MRI, diffusion weighted imaging(DWI). First, we found encephalomalacia and laminar
necrosis in the brain left parietotemporal lobe, hippocampus and bilateral frontal lobe with diffuse whitematter disorder (Fig. 1a). Second, cerebellar atrophy was
also diagnosed from the brain scan (Fig. 1b). No abnormality was identified in the craniocerebral MRA scan.
Clinical data of the family were collected and analyzed
under the approval by the Ethics Committee Xiangya

Whole exome sequencing (WES) was performed tosearch for potential pathogenic variants in an unbiased
way. Genomic DNA was extracted from peripheral bloodof the proband and his parents as previously described[8]. The putative mutations in Genomic DNA were subsequently screened by WES. Sequence variants werechecked with population databases gnomAD (http://gnomad.broadinstitute.org/) and evaluated using multiple
bioinformatic programs. Variant pathogenicity was interpreted according to the American College of Medical
Genetics (ACMG) guidelines [9]. The variant was further
confirmed by Sanger sequencing.
A novel variant c.524G > T (p.C175F) in folate receptor alpha (FOLR1) was identified. The Sanger sequencing
confirmed the homozygous state of the variant in the affected individual, with the parents of being the heterozygous carrier (Fig. 2a). The variant was absent from
population database gnomAD. Multiple bioinformatic
analysis results (Polyphen2 score 0.995, probably damaging; Mutation Taster score 0.998, disease causing; andSIFT score 0, deleterious) indicated that the variant wasdeleterious.
Further diagnostic assessment

Laboratory blood routine tests revealed the total folate
was 2.20 μg/L, lower than the minimal normal density of(3 μg/L) and the intracellular folate was 82.66 μg/L,
lower than minimal normal range (93 μg/L).
Further examination revealed that the patient had almost undetectable levels of CSF 5-MTHF. The average
concentration of 5-MTHF was 1.38 nmol/L, which isdramatically lower than the normal concentrations (60~ 210 nmol/L) for 6 ~ 15 years old children. The results
indicated the cerebral folate deficiency. Taken together,laboratory results and WES analysis demonstrated thatthe mutation in FOLR1 and therefore the CSF 5-MTHF
deficiency serves as the major etiology of patient’s
symptoms.
Treatment

We devised the following treatment procedures basedon genetic and laboratory test results: calcium folinate
was given to the patient by intravenous (iv) injection atdosage of 2 mg/kg/d for 1 week, and adjusted to 6 mg/
kg/d by oral administration, and gradually increased to
11 mg/kg/d orally at last. The CSF-5MTHF concentration was monitored closely during the treatment withcalcium folinate. We gradually reduced the supplementof antiepileptic drugs (AEDs) until the patient was seizure free at the end. After 2 months of treatment, theZhang et al. BMC Medical Genetics(2020) 21:235

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Fig. 1 Enhanced Brain MRI of a 6 years and 11 months old male patient. a MRI image of brain scan showed encephalomalacia and laminar
necrosis with diffuse white matter disorder. b MRI scan of the brain demonstrated cerebellar atrophy in the patient

CSF 5-MTHF concentration was elevated to 36.24 nmol/L, and 6 months later, the CSF 5-MTHF concentration
was increased to 78.76 nmol/L that fell into the normal
range of 60 ~ 210 nmol/L for 6 ~ 15 years old children.
Besides, the symptoms including unsteady gait, failure to
hold objects readily, epileptic seizure, etc. were considerably improved. Furthermore, after the treatment, seizure
no longer concomitantly happened with fever.

Conclusions and discussion
In this report, we documented a first case of CFD patient in China caused by a novel mutation of FOLR1

gene (Fig. 1a). The young patient had been transferred
between hospitals and the symptoms relapsed frequently
prior to the elucidation of the underlying cause of his
symptoms. The elaboration of the association between
the mutation of FOLR1 gene and the clinical manifestations greatly expedited the treatment of CFD in thiscase.
FOLR1 is glycophosphatidylinositol (GPI)-anchoredcell membrane protein, which regulates folate transport
into the cells [6]. Chen et al. studied the crystal structureof human FOLR1 in complex with folic acid at 2.8 Å
resolution [7]. Another group determined discreteFig. 2 Whole-exome sequencing (WES) and Sanger sequencing revealed missense variant of c.524G > T (p.C175F) in FOLR1 gene. a Homozygous
variant in proband and heterozygous variant in his parents were identified. b Evolutionary conservation of cysteine residue at position 175 (redbox) in the FOLR1 gene among species. wt, wildtype

Zhang et al. BMC Medical Genetics(2020) 21:235

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Fig. 3 Lollipop graph shows mutations in FOLR1 gene reported in literatures. Red star indicates the variant identified in this study. Green dots:
missense mutation; Red dots: inframe mutation; Blue dots: Truncating mutation. Green bar: Folate receptor family domain where folate binds.
Note: g.3576 T > G splice mutation (from ref. [8]) is not included in this graph due to its being out of the scope after re-annotation

structural conformations dependent on pH changes [10].
No direct evidence has been found to support that thenovel variant c.524G > T (p.C175F) is involved in ligand
binding. However, C175 is conserved among different
species, and clinical data shown in this study demonstrated the importance of this residue. Possibly, mutationof this site will somehow sabotage the protein function.
The further experiments will shed light on how this variant affects FOLR1 function as well as folate binding
ability.
Pathogenic variants attributed to neurological disorders have been recovered in FOLR1 gene locus [2, 5, 8,
10, 11]. Homozygous mutations or compound heterozygous mutations led to autosomal recessive disorders,
while a homozygous 18-bp in-frame duplication in
FOLR1 gene also linked to neurodegeneration [5]. It
transpires that patients carrying FOLR1 mutations exclusively end up with substantial low concentrations of CSF
5-MTHF (≤ 5 nmol/L), which lead to multiple brain
lesions as revealed by neuroimaging studies. By reviewing pertinent literatures, we summarized all existing 15
variants from 23 patients in Fig. 3 [2, 5, 8, 10, 12–17]. In
this case, we added a novel homozygous p.C175F variant
in FOLR1 genotype spectrum. Alignment of the protein
sequences of human FOLR1 with those from other organisms shows that the residue C175 is conserved acrossdifferent species (Fig. 2b), implying that this site is critical for normal protein function and mutant protein may
be defective in 5-MTHF binding and translocation. To
improve the low concentrations of 5-MTHF caused by
p.C175F variant, we deployed calcium folinate, the racemic stable form of folate, through oral or iv administration [18]. The treatment regime alleviated thesymptoms efficiently. Given that the overdose of folicacid treatment could cause neurotoxicity [19], it isimperative to distinguish folinic acid from folic acid, andthe latter may deteriorate the patient symptoms by
sequestration of folic receptors through tight binding. In
this respect, we meticulously monitored the changes in
CSF 5-MTHF concentrations to avoid any side effect of
the treatment.

In conclusion, the understanding of the genetic basisof frequently relapsed epileptic syndrome is critical fordevising an effective treatment. This could be a tremendous benefit to the young patient, because the earlier theeffective treatment is applied, the better recovery can be.
Abbreviations
CFD: Cerebral folate deficiency; 5-MTHF: 5-methyltetrahydrofolic acid;
CSF: Cerebrospinal fluid; FR: Folate receptor; MRI: Magnetic ResonanceImaging; WES: Whole Exome Sequencing; DWI: Diffusion weighted imaging;
ACMG: American College of Medical Genetics; AEDs: Antiepileptic drugs
Acknowledgements
We thank the patient and his family for the cooperation and contribution.
We would like to thank Dr. Xiaodong Wang from Cipher Gene, Inc. forassisting in manuscript editing.
Authors’ contributions
CZ, was the primary care physician of the patient. She analyzed the Sanger
sequencing data, conceptualized the paper and drafted the initialmanuscript and revised the paper. XD and YW helped to collect the clinicalinformation, followed up the patient and interpreted the data. FH evaluatedthe patient’s MRI. FY provided case consulting, treatment suggestions andrevised the paper. JP was responsible for analyzing the Sanger sequencing
data, drafting of the manuscript, carrying critical revision of the manuscript.
All authors have read and approved the manuscript.
Funding
This work was kindly supported by Hunan Provincial Natural Science
Foundation of China (No. 2020JJ5946) and National Natural Science
Foundation of China (No. 81801297; No. 81771408). Whole exome
sequencing was paid by funding money.
Availability of data and materials
The data that support the findings of this study are available from thecorresponding author upon reasonable request.
Data has been deposited and is publicly available in NCBI SRA database at
weblink: https://www.ncbi.nlm.nih.gov/Traces/study/?acc=PRJNA672777
Ethics approval and consent to participate
All procedures were performed in accordance with the ethical standards ofthe responsible Ethics Committee of Xiangya Hospital, Central South
University (Changsha, China) on human experimentation and with the
Declaration of Helsinki. For this work written informed consent was obtained
from all participants. For participants under 16 years old written informed
consent was obtained from the participant’s parent.
Consent for publication
Written informed consent was obtained from the parents for publication ofthis case report. Copy of the written consent is available for review by theeditor of this journal.

(2020) 21:235

Page 5 of 5Competing interests
None of the authors has any conflict of interest to disclose.

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Zhang et al. BMC Medical Genetics

Author details
1
Xiangya Hospital Central South University, 87 Xiangya Road, Changsha,
Hunan 410008, P.R. China. 2XiangYa School of Medicine of Central South
University, 172 Tongzipo Road, Changsha, Hunan 410013, P.R. China.
Received: 30 July 2020 Accepted: 3 November 2020

Publisher’s Note
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